1. Field of the Invention
The present invention relates to a monoclonal antibody, or a fragment thereof, for isolating and/or identifying at least one cell population which is selected from the group comprising haematopoietic stem cells, neuronal stem cells, neuronal pre-cursor cells, mesenchymal stem cells and mesenchymal precursor cells.
The term “stem cell” denotes, in a general manner, any cell which has not yet differentiated and which possesses the ability both to produce identical descendants and to differentiate into specific developmental lines.
Adult stem cells have the function of maintaining cell number homeostasis in the tissue concerned, i.e. of replacing cells which have died. For this reason, stem cells are particularly to be found in tissues which are subjected to high stresses. Adult stem cells have been found in a very wide variety of tissues and organs, such as, for example, bone marrow, brain, liver, skin, intestine, cornea, etc.
In the bone marrow, haematopoietic stem cells produce new cells continuously since these latter cells are constantly required in the blood owing to the limited life span of most of the cells.
The starting point for the formation of blood cells is the pluripotent, undifferentiated haematopoietic stem cell which is still not determined for a specific function. When stem cells differentiate, precursor cells, which are unable to replicate themselves and only bring a specialized cell type to maturity, are formed first of all. Neither the pluripotent stem cell nor the different intermediate stages are able to fulfil cell-specific haematopoietic functions; it is only the cells which have matured which are able to do this. Progenitor cells which have entered upon a particular differentiation route then also keep to this route until maturation is achieved (commitment).
In addition to stem cells for haematopoietic cells, stem cell-like cells which are progenitors of nonhaematopoietic tissues are also present in the bone marrow. These progenitors of non-haematopoietic tissues were originally termed, inter alia, tissue culture plastic-adherent cells and have more recently been termed either mesenchymal stem cells or bone marrow stroma cells (MSCs).
These cells are of interest not only because of their multipotency as regards differentiation; they are also of interest, for example, for their possible use in cell therapy and gene therapy.
The fact that, under certain conditions, mesenchymal stem cells can also differentiate into nerve cells means that, inter alia, there is a need to be able to distinguish these mesenchymal stem cells from neuronal progenitor cells.
These neuronal progenitor cells (termed NPCs below) are found in the central nervous system. They also express Nestin and are able to differentiate into neurones, astrocytes and oligodendrocytes.
Neuronal progenitor cells are CD133-positive; this cell surface marker was originally found on haematopoietic stem cells. However, it has recently been shown that this marker is also expressed by nervous tissue and skeletal muscle tissue. For these reasons, this marker is not suitable for distinguishing between different stem cells or progenitor cells on its own.
Since, as has been mentioned, haematopoietic stem cells continuously generate new cells in the bone marrow, stem cells coexist with the progenitor cells at the same time in the bone marrow. In the bone marrow, these cells are present in a complex arrangement, thereby making it difficult to identify rare cells. Stem cells and their direct descendants express a pheno-type which is virtually identical. For these reasons, it is not possible, either, to identify an ultimate stem cell simply on the basis of visible features.
The frequency of stem cells in the bone marrow is from 1×10−5 to 1×10−6. In addition, the stem cells are as a rule widely scattered in the given tissue, which means that they are difficult to detect.
As has been mentioned above, haematopoietic stem cells divide, under certain conditions, into progenitor cells whose further differentiation is to some degree already determined. Depending on the nature and quantity of the cytokines which are present, these myeloid and lymphoid progenitor cells can in turn generate a variety of other progenitor cells which are, however, no longer able to replicate themselves. Examples of cytokines which regulate haematopoiesis are granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), several interleukins, stem cell factor (SCF), erythropoietin (EPO), etc.
In order to investigate the haematopoietic (blood cell-forming) potential of stem cells, relevant human cell populations are transplanted into immunodeficient mice (NOD/SCID mice). If the transplanted cells are stem cells, it is then possible to detect human haematopoiesis in addition to the murine haematopoiesis. This in-vivo assay is used to characterize and identify stem cells by in fact analysing the progeny of individual cells.
2. Related Prior Art
As can be seen from the above, haematopoietic stem cells possess great therapeutic potential and are used in patients in whom the immune system is impaired or completely destroyed. FACS (fluorescence-activated cell sorter) can be used, for example, to 9-purify. haematopoietic stem cells from the bone marrow. This purification depends on the presence, on the stem cells, of particular cell surface proteins which distinguish the haematopoietic stem cells and the progenitor cells from other cell types and on the absence of other cell surface proteins, these latter proteins then being characteristic for differentiated haematopoietic cells. Each of the surface proteins binds a different monoclonal antibody, with each of these antibodies being conjugated to a different fluorescent dye, thereby making it possible to use FACS to separate the cells.
The cell surface marker CD34, in particular, has been used in the past for isolating haematopoietic stem cells.
In addition, antibodies directed against the antigen CD133 have recently been used for characterizing haematopoietic stem cells. Miraglia et al., “A novel five-transmembrane hematopoietic stem cell antigen: isolation, characterization and molecular cloning”, Blood 90: 5013-5021, (1997) have shown that this antigen is a 120 kDa glycoprotein which possesses five transmembrane domains and which is expressed not only on haematopoietic stem cells and their progenitors but also on neuronal and endothelial stem cells.
CD133 antibodies are used, in addition to the conventional CD34 antibodies, for positively selecting haematopoietic stem cells and progenitor cells on a clinical scale. CD133 is only expressed on CD34bright (high fluorescence intensity) stem cells and progenitor cells. CD34bright CD133-positive cells are in the main negative for other erythroid progenitor cell markers such as CD36 and glycophorin A. In addition to stem cells which induced human haematopoiesis in: the NOD/SCID mouse model, the majority of granulocyte/macrophage progenitor cells have also been found in CD133-positive fractions derived from human bone marrow and peripheral blood.